Synthesis of PHB by recombinant E. coli harboring an approximately 5 kb genomic DNA fragment from Streptomyces aureofaciens NRRL 2209

An approximately 5.0 kb Sau3A I genomic DNA fragment from Streptomyces aureofaciens NRRL 2209 was cloned in a plasmid vector and introduced into Escherichia coli. The recombinant E. coli accumulated polyhydroxyalkanoates (PHAs) as cytoplasmic inclusions. The accumulated PHA was identified as the isotactic homopolymer of PHB with a molecular weight of 2.85x10(5). Purified PHB granules were spherical with an average size of 1.1 microm and of stable configuration. DSC thermogram suggested high crystalline nature of the polymer. Maximum thermal degradation of the biopolymer occurred between 250 and 340 degrees C. Recombinant E. coli cells preferentially utilized glycerol as the carbon source and accumulated 25-28 times more PHB than the native S. aureofaciens.     

Screening and evaluation of probiotics as a biocontrol agent against pathogenic Vibrios in marine aquaculture

AIMS: The present work aims at finding potential probionts from marine sources as a biocontrol agent against pathogenic Vibrio species in shrimp larval culture. METHODS AND RESULTS: A total of 109 bacterial strains were isolated from seawater, sediment and marine fish-gut samples, and were screened for their antagonistic activity against Vibrio species. Three strains (Q, Q1 and M) isolated from the marine sediment were found antagonistic against Vibrio strains. Based on 16S ribosomal DNA gene sequence analysis, the strain Q was identified as Paenibacillus spp. (EF012164); Q1 as Bacillus cereus (DQ915582); and the M as Paenibacillus polymyxa (DQ915580). Further, the two bacterial species, Paenibacillus spp. and B. cereus were challenged separately at two different concentrations of 10(4) and 10(5) CFU ml(-1) for probiotic activity in the postlarvae of Penaeus monodon against pathogenic Vibrio harveyi and Vibrio spp. CONCLUSIONS: The present study identified the probiotic activity of Paenibacillus spp., B. cereus and Pa. polymyxa against the pathogenic Vibrios in the postlarvae of P. monodon. SIGNIFICANCE AND IMPACT OF THE STUDY: In vivo study reveals that the marine bacterial species can be used as probionts against pathogenic Vibrios in shrimp larval culture practices.     

A survey of the accumulation of novel polyhydroxyalkanoates by bacteria

Summary A wide range of bacterial strains were examined for their ability to accumulate polyhydroxyalkanoates (PHA) from various carbon sources. Strains were selected from those reported to accumulate poly-3-hydroxybutyrate (PHB), related organisms and laboratory stocks. Other strains known to utilize n-alkanes, n-alcohols or n-acids were chosen to investigate their ability to produce long-chain PHAs. Five strains accumulated only PHB, 13 accumulated PHAs containing only C₄ and C₅ units and 7 accumulated PHAs containing 3-hydroxyacid units in the range C₅ to C₁₀.     

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

AIMS: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. METHODS AND RESULTS: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. CONCLUSIONS: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.     

Peroxisomal polyhydroxyalkanoate biosynthesis is a promising strategy for bioplastic production in high biomass crops

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers with diverse plastic‐like properties. PHA biosynthesis in transgenic plants is being developed as a way to reduce the cost and increase the sustainability of industrial PHA production. The homopolymer polyhydroxybutyrate (PHB) is the simplest form of these biodegradable polyesters. Plant peroxisomes contain the substrate molecules and necessary reducing power for PHB biosynthesis, but peroxisomal PHB production has not been explored in whole soil‐grown transgenic plants to date. We generated transgenic sugarcane (Saccharum sp.) with the three‐enzyme Ralstonia eutropha PHA biosynthetic pathway targeted to peroxisomes. We also introduced the pathway into Arabidopsis thaliana, as a model system for studying and manipulating peroxisomal PHB production. PHB, at levels up to 1.6%–1.8% dry weight, accumulated in sugarcane leaves and A. thaliana seedlings, respectively. In sugarcane, PHB accumulated throughout most leaf cell types in both peroxisomes and vacuoles. A small percentage of total polymer was also identified as the copolymer poly (3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) in both plant species. No obvious deleterious effect was observed on plant growth because of peroxisomal PHA biosynthesis at these levels. This study highlights how using peroxisomal metabolism for PHA biosynthesis could significantly contribute to reaching commercial production levels of PHAs in crop plants.     

Prevalence of tet(B) and tet(M) genes among tetracycline-resistant Vibrio spp. in the aquatic environments of Korea

Of 24 tetracycline(Tc)-resistant Vibrio spp. isolated from different marine sources in Korea between 1993 and 2003, 23 were identified as carrying both tet(B) and tet(M), while 1 strain carried tet(B) only. In conjugation experiments, 3 strains appeared to be able to transfer both tet(B) and tet(M) to the recipient. Both discriminatory PCR and sequence analysis showed that tet(M) genes of Vibrio spp. appear to be a single allele containing a specific region of tet(M) in Tn1545. However, erm(B) and aphA3, known to be linked to Tn1545-like genes, were not detected in Tc-resistant Vibrio spp., even in 9 strains resistant to erythromycin. In analysis to examine the relative position of tet(B) and tet(M), it was shown that tet(M) was present at the 3'-end of the insertion sequence IS10 of Tn10 carrying tet(B). At the junctional region between Tn10 and tet(M), we found a 14 bp sequence of unknown function and the deletion of regulatory sequences reported to be needed for tet(M) expression in conjugative transposons. This is the first report of the simultaneous presence of tet(B) and tet(M), and of the tet(M) gene being linked to the 3'-end of Tn10 in Tc-resistant Vibrio spp. in Korea.     

High Prevalence of Vibrio cholerae Non-O1 Carrying Heat-Stable-Enterotoxin-Encoding Genes among Vibrio Isolates from a Temperate-Climate River Basin of Central Italy

Vibrio spp. of clinical interest from the Arno River basin (Tuscany, Italy) were investigated in this study. Vibrios were isolated from 70% of water samples. Vibrio cholerae non-O1 was the most prevalent species (82% of isolates), followed by Vibrio mimicus (10%) and Vibrio metschnikovii (8%). Recovery of vibrios was correlated with temperature, pH, and various indicators of municipal pollution. None of the 150 Vibrio isolates carried ctx-related genomic sequences, whereas 18 (14.6%) of the 123 V. cholerae non-O1 isolates and 1 (6.7%) of the 15 V. mimicus isolates carried sto alleles. These findings indicate that considerable circulation of sto-positive vibrios may occur in temperate-climate freshwater environments.     

Isolation of polyhydroxyalkanoate-producing bacteria from an integrated-farming pond and palm-oil mill effluent ponds

Bacterial isolates from two environments, an integrated-farming pond in the university and palm-oil mill effluent (POME) ponds at a local palm-oil-processing factory, were screened for polyhydroxyalkanoates (PHAs). Initially Sudan Black B staining was performed to detect lipid cellular inclusions. Lipid-positive isolates were then grown in a nitrogen-limiting medium containing 2% (w/v) glucose to promote accumulation of PHA before the subsequent Nile Blue A staining. The PHA extracted from positive isolates was confirmed by nuclear magnetic resonance (NMR) spectroscopy. The proportion of PHA-positive bacterial isolates was higher in the POME ponds compared to the integrated-farming pond.     

Rapid serological identification of Vibrio vulnificus by anti-H coagglutination

Staphylococcus aureus Cowan 1 cells were armed with anti-flagellar (anti-H) antibody produced in rabbits immunized with flagellar core protein prepared from Vibrio vulnificus. This reagent was assessed by coagglutination for its capacity to agglutinate and identify V. vulnificus. A species-specific H antigen is expressed in the core proteins of the polar flagella of V. vulnificus. Of 435 V. vulnificus isolates identified bacteriologically, 432 (99.3%) were agglutinated in the slide test within 2 min after the addition of the anti-V. vulnificus H coagglutination reagent. Other than Vibrio pelagius, the reagent did not agglutinate 19 heterologous Vibrio spp. tested, including 290 V. cholerae, 22 V. mimicus, 395 V. parahaemolyticus, and 16 V. fluvialis isolates recovered from seafood and the marine environment. The serological resolution of the coagglutination reaction was enhanced if the organism under test was suspended in 0.1 M Tris buffer-0.1 mM EDTA-1.0% Triton X-100 (TET) for 24 h before serological examination. The TET buffer also increased the sensitivity of the coagglutination reaction 100-fold over that for isolates suspended in 0.3% formalinized phosphate-buffered saline before testing. The anti-H coagglutination test is a rapid, serologically specific, and inexpensive procedure for identifying V. vulnificus one step beyond primary isolation.     

Rapid serological identification of Vibrio vulnificus by anti-H coagglutination.

Staphylococcus aureus Cowan 1 cells were armed with anti-flagellar (anti-H) antibody produced in rabbits immunized with flagellar core protein prepared from Vibrio vulnificus. This reagent was assessed by coagglutination for its capacity to agglutinate and identify V. vulnificus. A species-specific H antigen is expressed in the core proteins of the polar flagella of V. vulnificus. Of 435 V. vulnificus isolates identified bacteriologically, 432 (99.3%) were agglutinated in the slide test within 2 min after the addition of the anti-V. vulnificus H coagglutination reagent. Other than Vibrio pelagius, the reagent did not agglutinate 19 heterologous Vibrio spp. tested, including 290 V. cholerae, 22 V. mimicus, 395 V. parahaemolyticus, and 16 V. fluvialis isolates recovered from seafood and the marine environment. The serological resolution of the coagglutination reaction was enhanced if the organism under test was suspended in 0.1 M Tris buffer-0.1 mM EDTA-1.0% Triton X-100 (TET) for 24 h before serological examination. The TET buffer also increased the sensitivity of the coagglutination reaction 100-fold over that for isolates suspended in 0.3% formalinized phosphate-buffered saline before testing. The anti-H coagglutination test is a rapid, serologically specific, and inexpensive procedure for identifying V. vulnificus one step beyond primary isolation.     

Occurrence of pathogenic vibrios in coastal areas of France

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.     

Distribution, genetic richness and phage sensitivity of Vibrio spp. from coastal British Columbia

This study examined the distribution, susceptibility to viral infection and genetic diversity of Vibrio spp. in the coastal waters and sediments of British Columbia during summer (July and August). Abundances of presumptive Vibrio spp. ranged from 1.5 to 346 ml(-1) within the water column (1-291 m); whereas, abundances at the water-sediment interface were much higher (up to approximately 3 x 10(4)Vibrio spp. cc(-1)), and decreased with sediment depth (down to 30 cm). The genetic diversity of Vibrio spp. isolates was not tied to the location from which they originated and was only influenced in a minor way by the type of environment. However, the environment had a greater effect on phage-typing patterns. Vibrio parahaemolyticus isolates from environments with high abundances of cells (sediments and oysters) were generally more susceptible to viral infection than those from the water column which were highly resistant. Therefore, although Vibrio spp. were widespread in the areas investigated, the results show that there is segregation of bacterial host strains in different environments, under differing selection pressures, which ultimately will affect in situ phage production.     

Isolation and characterization ofVibrio vulnificus inhabiting the marine environment of the southwestern area of Taiwan

Vibrio vulnificus, a marine bacterium, is of concern in Taiwan because it causes wound infections and sepsis with a high mortality rate every year. To examine forV. vulnificus, 13 samples of seawater or oysters were collected from nine sites in Yunlin, Chiayi, and Tainan. Seventy-seven strains ofV. vulnificus were isolated from 11 samples. Among these environmental isolates, 72 (91%) were indole-positive, a characteristic of biotype 1. The remaining five strains although indole-negative, a characteristic previously found exclusively in biotype 2 strains, were all ornithine decarboxylase- and mannitol-positive, which has never been reported for biotype 2 strains. Based on the overall biochemical reactions obtained using a commercial identification system, these indole-negative strains appeared to be more like biotype 1. Fifty-seven ribotypes were identified among these isolates, indicating the great genetic divergence in this species. Of the 30 environmental isolates tested, 17 (56.7%) exhibited virulence comparable to the clinical isolates in the mouse, implying that a high proportion of theV. vulnificus strains in the marine environments might be pathogenic to humans.     

Extracellular degradation of medium chain length poly(beta-hydroxyalkanoates) by Comamonas sp.

The PHA-degrading isolate, strain P37C, was enriched from residential compost for its ability to hydrolyze the medium chain length PHA, poly(beta-hydroxyoctanoate) (PHO). It was subsequently found to grow on a wide range of PHAs, including both short chain length and medium chain length PHAs. The isolate was identified as belonging to the genus Comamonas. Strain P37C formed clear zones on poly(beta-hydroxybutyrate) (PHB), (PHO) and poly(beta-hydroxyphenylvalerate) (PHPV) overlay plates. PHA clear zone tubes were prepared using seven different kinds of PHAs, ranging from PHB with four-carbon repeating units, to poly(beta-hydroxyoctanoate-co-beta-hydroxyundecanoate) (PHOU) with 8- and 11-carbon repeating units. There was a direct correlation between PHA side chain length and rate of hydrolysis of the PHAs. A series of PHOUs containing varying percentages of unsaturated bonds were used to make a series of epoxidized PHOUs (PHOEs) with varying percentages of epoxy functions. Results of clear zone tube assays showed that these functionalized PHAs were all biodegradable by strain P37C, and there was no apparent correlation between rate of biodegradation and the proportion of functional groups in the PHAs. Biodegradability of these PHAs was verified using respirometry and enzyme assays. Cell-free supernatants containing activity toward PHAs were prepared, and strain P37C was shown to synthesize at least two distinct PHA depolymerases for the hydrolysis of SCL and MCL PHAs.     

Denaturing gradient gel electrophoresis analyses of the vertical distribution and diversity of Vibrio spp. populations in the Cariaco Basin

The Cariaco system is the second largest permanently anoxic marine water body in the world. Its water column is characterized by a pronounced vertical layering of microbial communities. The goal of our study was to investigate the vertical distribution and diversity of Vibrio spp. present in the Cariaco Basin waters using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments. Representatives of the Vibrio genus were detected by nested and direct PCR in seawater at 10 depths. Sequence analyses of 55 DGGE bands revealed that only 11 different operational taxonomic units (OTU) are identified as Vibrio species. Between one and five OTUs were detected at each depth and the most common OTUs were OTU 1 and OTU 2, which phylogenetically clustered with Vibrio chagasii and Vibrio fortis, respectively. OTUs 3 and 4 were only found in the anoxic zone and were identified as Vibrio orientalis and Vibrio neptunius, respectively. Several Vibrio species detected are potentially pathogenic to human, prawns and corals such as Vibrio parahaemolyticus, Vibrio fischeri and Vibrio shilonii. In the Cariaco Basin, different Vibrio species were found to be specific to specific depths strata, suggesting that this genus is a natural component of the microbial communities in this marine redox environment.     

Accumulation of Poly (3-Hydroxybutyric Acid) by Some Soil Streptomyces

In a limited-scale survey, 55 soil streptomycetes were screened for the accumulation of poly (3-hydroxybutyrate) [PHB]. Only 18% of the isolates accumulated PHB ranging between 1.9–7.8% of the dry biomass. The promising isolate DBCC-719, identified as Streptomyces griseorubiginosus, accumulated PHB amounting to 9.5% of the mycelial dry mass in the early stationary phase when grown in chemically defined medium with 2% (wt/vol) glucose as the sole source of carbon. Nitrogen-limiting conditions were inhibitory to growth and PHB accumulation. The isolated polymer was highly soluble in chloroform, gave a sharp peak at 235 nm on digestion with concentrated H₂SO₄, and had a characteristic infrared spectrum. Received: 26 March 1999 / Accepted: 3 May 1999     

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol

Aims: To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. Methods and Results: Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram‐negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98·5% and 96·2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41°C and pH 7·3 and accumulated PHB up to 80·4% (w/w) of cell dry weight. PHB accumulation was growth‐associated. Although it was not an absolute requirement, 20 g l^?1 sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. Conclusions: A new PHB‐accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. Significance and Impact of the Study: This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.     

Poly-beta-hydroxybutyrate-accumulating bacteria protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii

A poly-beta-hydroxybutyrate (PHB)-accumulating enrichment culture was obtained using activated sludge from a polyphosphate-accumulating reactor as inoculum. PHB accumulated by the enrichment culture significantly enhanced the survival of Artemia nauplii, infected with the virulent pathogen Vibrio campbellii LMG 21363. A strain was isolated from the enrichment culture, based on its ability to accumulate PHB, and 16S rRNA gene sequencing of the isolate revealed 99% sequence similarity to Brachymonas denitrificans AS-P1. The isolate, named PHB2, showed good PHB-accumulating activity (up to 32% of the cell dry weight). PHB accumulated by isolate PHB2 was able to protect Artemia completely from the V. campbellii strain. Our data indicate that PHB-accumulating bacteria, such as B. denitrificans PHB2, could be used as an an effective and economically interesting alternative strategy to control infections in aquaculture.     

钩端螺旋体聚Beta羟基丁酸(PHB)生物合成途径分析

【目的】致病型问号钩端螺旋体(问号钩体, Leptospira interrogans)和腐生型双曲钩体(L. biflexa)能够大量合成菌体内贮藏物, 这可能是钩体在营养贫瘠环境中长时间存活的主要原因之一。本研究对钩体聚Beta羟基丁酸(PHB)贮藏物进行定性定量测定, 通过基因组分析补充定义PHB合成主要功能基因, 并采用分子生物学方法初步证明PHB合成途径的完整性, 为进一步研究PHB合成与钩体抗逆能力的关系奠定基础。【方法】采用脂类特异性尼罗红染色法和浓硫酸氧化-紫外分光光度计测定法, 对问号钩体和双曲钩体的PHB贮藏物进行定性定量测定; 采用生物信息学方法(BLAST和InterProscan/InterPro2Go), 通过同源性分析和功能结构域搜索寻找钩体基因组中的PHB合成相关基因; 最后采用克隆测序和定量RT-PCR技术检测相关基因表达情况, 初步验证生物信息学预测结果。【结果】尼罗红染色和氧化后比色定量实验证明钩体合成细菌常见贮藏物PHB, 问号钩体合成量为菌体干重的42%?45%, 双曲钩体合成量为64%?68%。尽管已公布的多个钩体基因组中均没有定义完整的PHB合成途径, 但本研究通过综合生物信息学分析, 在问号钩体和双曲钩体中鉴定了PHB合成途径的主要功能基因(phbC)。克隆测序和定量RT-PCR证实钩体转录表达大部分PHB合成相关基因(phbA/B/C), 说明钩体内该生物途径基本完整, 且部分高水平表达基因可能是钩体主要的PHB合成相关基因。【结论】问号钩体和双曲钩体均可合成PHB贮藏物, 且具有基本完整的PHB合成生物途径。     

Vibrios isolated from the cultured manila clam (Ruditapes philippinarum): numerical taxonomy and antibacterial activities

AIMS: A numerical taxonomic study of halophilic Vibrio isolated from healthy and brown ring disease (BRD) affected manila clams (Ruditapes philippinarum), harvested from the Atlantic coast of south-western Spain, was performed. METHODS AND RESULTS: Characterization of 123 presumptive Vibrio spp. was carried out using 94 phenotypic tests. Simple matching and Jaccard similarity coefficients were used for numerical analysis. Cluster analysis by the unweighted pair group method with arithmetic averages yielded 15 phena defined at 0.81 similarity. Large phena corresponded to Vibrio tubiashii, V. splendidus biotype I and V. harveyi (phena 1, 5 and 9, respectively). The species V.splendidus biotype II, V. natriegens, V. mediterranei and V. alginolyticus were also represented. The inhibitory effect of diffusible extracellular products of the isolates against 27 strains of V.tapetis, the aetiological agent of BRD, was also investigated. Only five V. tubiashii isolates inhibited the growth of V. tapetis strains. The antimicrobial effect was inhibited by heating and depended on the culture medium. CONCLUSIONS: The main Vibrio species associated with manila clams were V. tubiashii, V.spendidus and V. harveyi. The antagonistic relationship established between V. tapetis and the Vibrio spp. clam microbiota may explain the failure of isolation in plating medium of V.tapetis from BRD-affected clams on the south Atlantic coast of Spain. SIGNIFICANCE AND IMPACT OF THE STUDY: Some of the strains isolated from manila clams correspond to agarolytic strains that constitute phenon 7 and they do not fit into any of the currently described Vibrio species.